Gold nanoparticle-loaded macrophages enhance radiotherapy via immune remodeling in oral cancer

Abstract

Gold nanoparticles (GNPs) are promising radiosensitizers owing to their high atomic number, but their therapeutic efficacy is often limited by reticuloendothelial system (RES)-mediated clearance and poor tumor accumulation attributable to elevated interstitial fluid pressure. This study explored macrophage-mediated GNP delivery to enhance radiotherapy (RT) efficacy in oral cancer by leveraging the tumor-homing ability, immune plasticity, hypoxia tropism, and RES evasiveness of macrophages. Murine oral cancer cells (MCTQ1) and macrophages (RAW 264.7) were used. GNPs were synthesized using the Turkevich method, which were then assessed for radiosensitization using cell viability and colony formation assays. GNPs were radiolabeled with Iodine-131 (¹³¹I) using the chloramine-T method, and uptake by RAW 264.7 cells was quantified at various time points to optimize the generation of GNP-loaded macrophages (GNP@Rs). MTCQ1 tumor-bearing mice were divided into control, RT, GNP, RAW, GNP + RT, RAW + RT, and GNP@R + RT groups. Tumor volumes were monitored after GNP or cell administration and/or RT (8 Gy). Flow cytometry was used to analyze the immune cell populations post-treatment. Transwell assays confirmed that GNP loading did not impair macrophage migration, and in vivo fluorescence imaging demonstrated strong tumor tropism of RAW 264.7 cells and GNP@Rs. GNP@Rs maintained their migration ability and exhibited robust tumor accumulation for up to 96 h. Notably, GNP@R + RT treatment significantly enhanced tumor suppression relative to RT alone and increased the infiltration of macrophages, activated dendritic cells, CD4⁺ and CD8⁺ T cells, and natural killer cells. Macrophage-mediated GNP delivery successfully improved RT outcomes in oral cancer by increasing radiosensitivity and modulating immune microenvironment.

Keywords: Gold nanoparticles; Immune response; Macrophage-based delivery; Radiosensitization; Radiotherapy.

Graphical abstract

Fig. 1

40 nm gold nanoparticles (GNPs) enhance cancer cell radiosensitivity more effectively than 20 nm GNPs. (A–C) Representative Transmission Electron Microscopy (TEM) images of GNPs synthesized using citric acid-to-HAuCl₄ molar ratios of 2.0, 2.4, and 2.8, respectively. Scale bar = 50 nm. (D) Quantitative measurement of GNP diameters obtained from TEM images. (E–H) Cell viability of MTCQ1 and E0771 cells treated with indicated concentrations of 20 nm or 40 nm GNPs, combined with 0, 4, or 8 Gy X-ray radiation. (I–L) Combination index (CI) values calculated from cell viability results for the combined treatments in MTCQ1 and E0771 cells. Data in panels D–L are presented as mean ± SEM from three independent biological replicates. Statistical significance for relevant comparisons was determined using One-way ANOVA: ∗P < 0.05; ∗∗P < 0.01, ∗∗∗∗P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Preparation and cellular uptake of radiolabeled GNPs. (A) Schematic illustrating the conjugation of Iodine-131 (¹³¹I) to GNPs. (B–C) Representative radio-thin layer chromatography (radio-TLC) images verifying the radiolabeling purity of crude and purified ¹³¹I-labeled GNPs (¹³¹I-GNPs). (D) Stability assessment of ¹³¹I-GNPs over time in normal saline and FBS. (E–F) Quantified cellular uptake of ¹³¹I-labeled 20 nm GNPs and 40 nm GNPs by RAW 264.7 macrophages after incubation for a specified time. Quantitative data in panels D–F are presented as mean ± SEM from three independent biological replicates. Statistical significance for relevant comparisons was determined using One-way ANOVA: ∗P < 0.05; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 3

GNPs-loaded RAW264.7 cells exhibit polarization towards an M1 phenotype. Untreated RAW 264.7 cells served as the negative control (CTRL) for comparison. Lipopolysaccharide (LPS, 1 μg/mL) and Interleukin-4 (IL-4, 20 ng/mL) served as positive controls to induce M1 and M2 polarization, respectively. Representative bright-field microscopy images of RAW 264.7 cells (A) and GNP@Rs (B). Scale bar = 10 μm. Relative mRNA levels of M1 markers (Nos2, Tnf) and M2 marker (Arg1) in CTRL, LPS-treated, IL-4-treated, and GNP@R cells, assessed by qPCR after 6 h (C) and 24 h (D) of incubation. (E) Representative flow cytometry histograms displaying expression of iNOS (M1 marker) and Arg-1 (M2 marker) in the different treatment groups at 6 and 24 h. Black lines indicate fluorescence intensity gating relative to isotype controls. (F) Ratio of iNOS to Arg-1 determined by flow cytometry for all treatment groups at 6 and 24 h. Data shown in panels C, D, and F are presented as mean ± SEM from three independent biological replicates. Statistical significance for relevant comparisons was determined using One-way ANOVA: ∗P < 0.05; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4

Comparable in vitro and in vivo migration ability of RAW 264.7 cells and GNP@Rs. Representative images from Transwell assays showing migration of RAW 264.7 cells and GNP@Rs towards (A) standard medium or (B) MTCQ1-conditioned medium (CM). (C) Quantification of migrated cells from the in vitro assays depicted in panels A and B. Data represent mean ± SEM from three independent experiments. Representative in vivo fluorescence images of MTCQ1 tumor-bearing mice at 48 and 96 h following intravenous injection of fluorescence-labeled RAW 264.7 cells (D), and (E) the corresponding ex vivo fluorescence images of major organs (spleen, tumor, lungs, liver, kidneys, pancreas). (F) Representative in vivo fluorescence images of MTCQ1 tumor-bearing mice at 48 and 96 h following intravenous injection of fluorescence-labeled GNP@Rs, and (G) the corresponding ex vivo fluorescence images of major organs. Fluorescence signal intensity unit for in vivo and ex vivo imaging (D–G) is photons/second/cm²/steradian (ph/s/cm²/sr). SP, spleen; TM, tumor; LG, lung; LV, liver; PC, pancreas; K, kidney; H, heart.

Fig. 5

Combination therapy of GNP@Rs and RT enhances tumor control and modulates the tumor immune microenvironment in MTCQ1 tumor-bearing mice. (A) Schematic diagram illustrating the experimental timeline and treatment administration protocol. (B) Tumor growth curves for the different treatment groups (n = 6 mice per group). Data points represent mean tumor volume ±SEM. Statistical analysis performed using Two-way ANOVA. ∗, P < 0.05; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001. Flow cytometric analysis of tumor immune cell populations (n = 4 mice per group). Panels display the frequency of: (C) CD11b⁺F4/80⁺ macrophages, (D) CD11c⁺CD86⁺ activated dendritic cells (DCs), (E) CD4⁺ helper T cells, (F) CD8⁺ cytotoxic T cells, and (G) CD45⁺NK1.1⁺ Natural Killer (NK) cells. Data are presented as mean ± SEM. Statistical analysis performed using One-way ANOVA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.

Fig. 6

Macrophage-based GNP delivery combined with RT modulates immune cell populations in sentinel lymph nodes (SLNs). Frequency of immune cell populations in SLNs: (A) CD11b⁺F4/80⁺ macrophages, (B) CD11c⁺CD86⁺ activated DCs, (C) CD4⁺ T cells, (D) CD8⁺ T cells, and (E) CD45⁺NK1.1 NK cells in lymph nodes. Data are presented as mean ± SEM. Statistical analysis performed using One-way ANOVA. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Fig. 7

Hemolysis assay indicates a low hemolytic potential of RAW 264.7 cells and GNP@Rs. (A) Visual assessment of hemolysis. Red blood cells were treated with PBS (negative control), 20 % Triton X-100 (positive control), GNP, RAW 264.7 cells, and GNP@Rs. Undetectable hemolysis was observed in all experimental groups, except for positive control. Samples labeled (a) GNP, (b) RAW 264.7 cells, and (c) GNP@Rs represent a 1X dose equivalent used in vivo; samples (d) and (e) represent PBS and 20 % Triton X-100 controls, respectively. (B) Quantitative assessment of hemolysis at increasing doses (from 1X to 50X the in vivo equivalent). Data presented as mean ± SEM from three independent biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)